1. Technical Field
The present invention relates to the filed of biological technology, and more particularly to a stem cell culture method.
2. Related Art
Stem cells are a class of self-renewing pluripotent cells, which are able to be differentiated into many different functional cells under certain conditions. The stem cells may be classified by two methods. In a first method, the stem cells are classified into embryonic stem cells (ES cells) and somatic stem cells according to the development stage of the stem cells. In a second method, the stem cells are classified into totipotent stem cells (TSCs), pluripotent stem cells, and unipotent stem cells according to the development potential of the stem cells.
ES cells are mainly derived from inner cell mass (ICM) of a blastocyst and early embryonic cells before a fertilized ovum is developed into a morula. Human ES cells have 2 distinguished features, one is the high in-vitro self-renewing ability, and the second one is the ability to be differentiated into various types of cells in human body by directed induction.
Induced pluripotent stem cells (also referred to as iPSs) are ES cell-like pluripotent cells obtained by directly reprogramming animal or human somatic cells by introducing some factors into the somatic cells through gene transfection technology. This type of cells are very similar to the ES cells, as far as the cell morphology, growth characteristics, surface markers, and teratoma formation are concerned (Okita K, Ichisaka T, Yamanaka S. Nature 2007; 448: 313-317; Wernig M, Meissner A, et al. Nature 2007; 448: 318-324; Takahashi K, Tanabe K, Ohnuki M, et al. Cell 2007; 131: 861-872; and Yu J, Vodyanik M A, Smuga-Otto K, et al. Science 2007; 318: 1917-1920).
Somatic stem cells include stem cells found in adult tissues, such as bone marrow, umbilical cord or peripheral blood hematopoietic stem cells, bone marrow or umbilical cord mesenchymal stem cells, epidermal stem cells, adipose-derived stem cells, pancreatic stem cells, and neural stem cells, and can be theoretically differentiated into specific tissues and organs under specific conditions, thus being a foundation for repair and regeneration.
The stem cells have a wide application prospect in medicine. However, if the stem cells are intended to be successfully used in clinical practice, how to maintain the stem cells in an undifferentiated state while the stem cells proliferate in vitro, i.e., self renewal, is a first subject needed to be solved. The in-vitro culture of the stem cells requires 2 basic conditions, that is, the differentiation of the cells is inhibited while the division and proliferation of the cells are facilitated. In an existing stem cell culture technology, feeder layer cells and/or cell factors are generally used to meet the above conditions. For the ES cells, the commonly used feeder layer cells include, for example, murine embryonic fibroblasts (MEFs) and STO cells, which lose the division ability after, for example, irradiation with γ ray or treatment with mitomycin C. The cells can still survive and have the ability to assimilate the culture medium while losing the division ability. Use of the MEFs as the feeder layer to culture the ES cells is the earliest and most commonly used method [Takahama Y, Ochiya T, Sasaki H, et al. J. Oncogene, 1998, 16(24):3189-3196]; and the culture medium is also supplemented with cell differentiation inhibitors such as leukemia inhibitory factor (LIF) [Horak V, Flechon J E. Reprod Nutr Dev, 1998, 38(6):683-695]. However, the disadvantages in use of the MEFs to culture the ES cells are that (1) in culturing and expanding the human ES cells, the MEFs may spread animal pathogens to the human ES cells through the culture medium; and (2) a large amount of MEFs need to be produced, since the MEFs have a limited life cycle, cannot be passaged in vitro for a long period of time, and have an ability of generating the proliferation factor and the differentiation inhibitor that is diminished or even lost with the extension of the passage time; (3) it is difficult to obtain pure ES cells for biochemical and molecular biological analysis; and (4) the release of the chromosome from dead MEFs may cause the mutation of the ES cells and affects the maintenance of normal karyotype. In view of the problems brought by use of the MEFs in clinical, some scholars establish a culture medium with human source embryonic or somatic cells as the feeder cell and without animal-derived ingredients. The long-term undifferentiated proliferation state of the human ES cells can be maintained by respectively using human embryonic fibroblasts (HEFs), adult oviductal epithelial cells, human bone marrow stromal cells, or human foreskin cells as the feeder layer in place of the MEFs [Richards M, Fong C Y, Chan W K, et al. Nat Biotechnol, 2002, 20 (9):933-936; Cheng L, Hammond H, Ye Z, et al. Stem Cells, 2003, 21(2):131-142; and Meng G, Liu S, Krawetz R, et al. Stem Cells Dev. 2008, 17(3):413-22]. However, the culture system based on human feeder layer still requires the feeder layer cells and the ES cells to grow at the same time, and the human tissues cannot meet the demand for in-vitro culture and expansion of the ES cells due to the difficulty exiting in obtaining of the human tissues. In order to solve the difficulty exiting in obtaining of the human tissues, human placenta tissue is used and cells are isolated therefrom to culture embryonic stem cells. Genbacev et al [Genbacev O., Krtolica A., Zdravkovic T., et al. Fertil Steril 2005, 83, 1517-1529.] isolate human placenta fibroblasts from aborted pregnancy tissue of 6-9 weeks, and find that the human placenta fibroblasts can support the growth of the human embryonic stem cells and keep them in the undifferentiated state. However, the treatment through which the feeder layer cells lose the division ability, for example, irradiation with γ ray or treatment with mitomycin C, increases not only the operation steps, but also potential risks, because mitomycin C is a DNA inhibitor, which may cause the chromosome deformity of the ES cells.
For this reason, in order to overcome the problem of contamination caused by animal-derived ingredients, attempts are made to use a serum-free and feeder layer-free system to culture the stem cells, in which various growth factors are supplemented to ensure that the stem cells are maintained in the undifferentiated sate while continuously proliferating in vitro. However, the growth factors and the culture medium containing the same are very expensive, thereby increase the research and application cost.